Defining the molecular interactions between a virus and its host that regulate gene-specific transactivation has been essential to understanding DNA virus persistence and replication. The Human herpesvirus-8 (HHV-8) ORF50/Rta protein is necessary and sufficient for the virus to emerge from latency and replicate (lytic reactivation). Rta interacts directly with the cellular protein called RBP-Jk, which is also required for lytic reactivation. RBP-Jk normally specifies the genes that will be activated by the cellular Notch signal transduction pathway by binding sequence specifically to DNA. In this fashion, RBP-Jk serves as a landing pad for the activated Notch receptor (Notch intracellular domain (NICD)). HHV-8 Rta promotes DNA binding of RBP-Jk during viral reactivation, a mechanism that is fundamentally different from that established for other RBP-Jk-activating proteins. Our preliminary data suggest a gene-specific mechanism for controlling RBP-Jk-dependent activity in HHV-8 infected cells. The overall goal of this application is to define the basic molecular mechanisms that regulate RBP-Jk dependent-transactivation in HHV-8 infected cells. Our studies will shed light on the fundamental regulation of productive and non-productive virus reactivation as determined by promoter-specific transactivation. We will therefore comprehensively identify proteins that bind to essential HHV-8 promoters (Aim 1). We will define the molecular interactions required for Rta stimulation of RBP-Jk DNA binding (Aim 2). We will determine the molecular requirements for forming functional RBP-Jk-containing promoter complexes and HHV-8 reactivation (Aim 3). A series of biochemical and molecular biological approaches are proposed. A biochemical screen for promoter-specific protein-DNA interactions is a major approach. Promoter-reporter, protein-protein and protein-DNA interactions represent the basis for many of the experiments, and include novel, highly quantitative in vitro and in vivo assays. This proposal will shed light on how Notch target genes are specified, and reveal new components of the Notch signal transduction pathway. PUBLIC HEALTH RELEVANCE: This project will advance scientific understanding of the mechanisms by which Human herpesvirus-8 (HHV- 8) interacts with Humans to cause disease. Specifically, the experiments will reveal mechanisms by which the key viral protein Rta re-specifies the target genes stimulated by a cellular signaling pathway that has been associated with Human pathologies. Unique drug targets and diagnostic markers may be revealed.